The discovery that selenocysteine is encoded by UGA codons in both prokaryotic and eukaryotic proteins, and that UGA codons specifying both selenocysteine and termination are present in the same cells, led to the fundamental question: How is UGA encoding selenocysteine distinguished from UGA specifying termination? Because the UGA codons in mRNAs encoding selenoproteins are translated with fidelity, and those used as stop codons are efficiently recognized as such, there must exist features of the mRNAs themselves that allow the translational apparatus to distinguish between these two functions. UGA codon recognition in the eukaryotic selenoproteins, iodothyronine 5' deiodinase (5'DI) and glutathione peroxidase (GPX), requires putative stem-loop structures in the 3' untranslated regions of these mRNAs. Cellular factors, which may be protein, RNA, or a combination of the two, must somehow recognize these structures and facilitate binding of selenocysteyl-tRNA(UCA) to the ribosome. The purpose of the proposed studies is to identify and characterize the cellular components responsible for this recognition process, and the specific features which they recognize, with the long- term goal of understanding the mechanism of this critical process. Initial studies will focus on further characterizing mRNA sequences and secondary structures required for selenocysteine insertion. The cellular factors which interact with these sequences or structures will then be studied using RNA-protein gel mobility shift assays, UV crosslinking, and avidin biotin-RNA affinity chromatography. The selenocysteine insertion RNA sequences will be used as probes to screen expression libraries for proteins which specifically bind to them, following methods developed for cloning of sequence-specific DNA-binding proteins. Selenium deficiency is associated with an often fatal cardiomyopathy in China, Keshan disease. Selenocysteine is required for optimal conversion of thyroxine to 3,5,3'-triiodothyronine, the first step in thyroid hormone action. High levels of selenium have been found to produce developmental disorders in birds. The effects of selenium deficiency and toxicity in man and animals underscore the importance of this essential trace element.